This application is directed to methods and external guide sequence compositions designed to target cleavage of RNA by RNAse P.
I. Ribozyines and External Guide Sequence Molecules
Ribonucleic acid (RNA) molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, but also as enzymes which specifically cleave nucleic acid molecules. Such catalytic RNA molecules are called ribozymes.
The discovery of catalytic RNA, by Drs. Altman and Cech, who were awarded the Nobel prize in 1989, has generated much interest in commercial applications, particularly in therapeutics (Altman, Proc. Natl. Acad. Sci. USA 90:10898-10900 (1993); Symons, Annu. Rev. Biochem. 61:641-671 (1992); Rossi et al., Antisense Res. Dev., 1:285-288 (1991); Cech, Annu. Rev. Biochem. 59:543-568, (1990)). Several classes of catalytic RNAs (ribozymes) have been described, including intron-derived ribozymes (WO 88/04300; see also, Cech, T., Annu. Rev. Biochem., 59:543-568, (1990)), hammerhead ribozymes (WO 89/05852 and EP 321021 by GeneShears), axehead ribozymes (WO 91/04319 and WO 91/04324 by Innovir).
RNAse P
Another class of ribozymes include the RNA portion of an enzyme, RNAse P, which is involved in the processing of transfer RNA (tRNA), a common cellular component of the protein synthesis machinery. Bacterial RNAse P includes two components, a protein (C5) and an RNA (M1). Sidney Altman and his coworkers demonstrated that the M1 RNA is capable of functioning just like the complete enzyme, showing that in Escherichia coli the RNA is essentially the catalytic component, (Guerrier-Takada et al., Cell 35:849-857 (1983)). In subsequent work, Dr. Altman and colleagues developed a method for converting virtually any RNA sequence into a substrate for bacterial RNAse P by using an external guide sequence (EGS), having at its 5' terminus at least seven nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 5' terminus the nucleotides NCCA (N is any nucleotide)(WO 92/03566 and Forster and Altman, Science 238:407-409 (1990)). Using similar principles, EGS/RNAse P-directed cleavage of RNA has been developed for use in eukaryotic systems, (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992)). As used herein, "external guide sequence" and "EGS" refer to any oligonucleotide that forms an active cleavage site for RNAse P in a target RNA.
II. Hepatitis B Virus (HBV)
HBV, a member of a group of small DNA-containing viruses that cause persistent noncytopathic infections of the liver, is an infectious agent of humans that is found worldwide and which is perpetuated among humans in a large reservoir of chronic carriers. It is estimated that about 6-7% of the earth's population is infected (300 million carriers). The prevalence of the infection is not uniform throughout the world. There is a geographic gradient in distribution of HBV. It is lowest in North America and Western Europe, where the virus can be detected in 0.1 to 0.5% of the population, and highest in Southeast Asia and sub-Saharan Africa, where the frequency of infection may vary from 5 to 20% of the population. This skewed distribution parallels that of hepatocellular carcinoma and provides strong epidemiologic evidence for an association between chronic HBV infection and this type of malignancy.
Hepatitis B is of great medical importance because it is probably the most common cause of chronic liver disease, including hepatocellular carcinoma in humans. Infected hepatocytes continually secrete viral particles that accumulate to high levels in the blood. These particles are of two types: (i) noninfectious particles consisting of excess viral coat protein (HBsAg) and containing no nucleic acid (in concentrations of up to 10.sup.13 particles/ml blood), and (ii) infectious, DNA-containing particles (Dane particles) consisting of a 27 nm nucleocapsid core (HBcAg) around which is assembled an envelope containing the major viral coat protein, carbohydrate, and lipid, present in lower concentrations (10.sup.9 particles/ml blood). The human hepatitis B virus is a member of the Hepadna Viridae family, with close relatives including woodchuck hepatitis virus (WHV), duck hepatitis virus (DHV), and ground squirrel hepatitis virus (GHV) (Robinson (1990)). Like retroviruses, the hepadnavirus utilizes reverse transcription of its 3.2 kb DNA genome (Pugh (1990)). The genome of hepatitis B virus is circular and partially single-stranded, containing an incomplete plus strand. The incomplete plus strand is complexed with a DNA polymerase in the virion which has been shown to elongate the plus strand using the complete minus strand as the template. These morphological and structural features distinguish hepatitis B viruses from all known classes of DNA-containing viruses.
The replication cycle of hepatitis B viruses is also strikingly different from other DNA-containing viruses and suggests a close relationship with the RNA-containing retroviruses. The principal unusual feature is the use of an RNA copy of the genome as an intermediate in the replication of the DNA genome. Infecting DNA genomes are converted to a double-stranded form which serves as a template for transcription of RNA. Multiple RNA transcripts are synthesized from each infecting genome, which either have messenger function or DNA replicative function. The latter, termed "pre-genomes," are precursors of the progeny DNA genomes because they are assembled into nucleocapsid cores and reverse-transcribed into DNA before coating and export from the cell. Thus each mature virion contains a DNA copy of the RNA pre-genome and a DNA polymerase.
The first DNA to be synthesized is of minus strand polarity and is initiated at a unique site on the viral genetic map. Very small nascent DNA minus strands (less than 30 nucleotides) are covalently linked to a protein, and are likely to act as primer for minus strand DNA synthesis. Growth of the minus strand DNA is accompanied by a coordinate degradation of the pre-genome so that the product is a full-length single-stranded DNA, rather than an RNA:DNA hybrid. Plus strand DNA synthesis has been observed only after completion of the minus strand, and initiates at a unique site close to the 5' end of the minus strand. Complete elongation of the plus strand is not a requirement for coating and export of the nucleocapsid cores, thus most extracellular virions contain incomplete plus strands and a large single-stranded gap in their genomes. Because the hepatitis virus genome is autonomous and does not utilize a DNA-to-DNA pathway for its replication, continuous intracellular replication of its genome is essential for the maintenance of the virus.
The hepatitis B virus surface antigens (HBsAgs), which make up the viral envelope, are polypeptides encoded by the pre-S2, pre-S2 and S genes of the virus. The major protein is the 226 amino acid S gene product derived from a 2.1 kb subgenomic message.
III. Acute Promyelocytic Leukemia (APL)
About 10% of acute myeloblastic leukemias (AML) in adults is acute promyelocytic leukemia (APL, French American British Classification (FAB) M3), see Warrell et al., New England J. Med., 329:177-189 (1993) for reviews). The disease typically presents with a bleeding diathesis which is often exacerbated by chemotherapy, leading to a high rate of early mortality, primarily from intracranial hemorrhage. The bleeding diathesis is due to the presence of malignant promyelocytes which release procoagulant substances. These, in turn, activate the coagulation cascade, depleting fibrinogen, clotting factors and platelets.
While conventional chemotherapy can achieve complete remission in most patients, the five year survival averages only 35 to 45 percent. These figures do not include the high degree of early mortality (Warrell et al. (1993)).
A second avenue of therapy for APL patients involves the use of retinoids, in particular all-trans retinoic acid (ATRA; commercially available as TRETINOIN, Hoffman La Roche, Nutley, N.J.). In several published studies TRETINOIN has been able to induce remission in about 48% of the patients treated (Warrell et al. (1993); Huang et al., Blood, 72:567-572 (1988); Castaigne et al., Blood, 76:1704-1709 (1990); Warrell et al., New Engl. J. Med., 324:1385-1393 (1991); Cheson, New England J. Med., 327:422-424 (1992)). However, the duration of the remission is short, averaging 3.5 months, following which patients display an acquired resistance to the retinoid. This resistance is probably explained by an increased clearance of the drug from the bloodstream, due to the induction of cytochrome P-450 enzymes and increased expression of cellular retinoic acid-binding proteins. Combination of retinoid treatment with conventional chemotherapy is actively pursued at present, with initial results indicating a 60 to 70% cure (Cheson, New England J. Med., 327:422-424 (1992)).
APL is consistently associated with a non-random chromosomal abnormality, characterized by a balanced and reciprocal translocation between the long arms of chromosomes 15 and 17 (t(15;17)), found in over 90% of patient-derived APL cells (Kakizuka et al., Cell, 66:663-674, (1991); de The et al., Cell, 66:675-684 (1991); Pandolfi et al., Oncogene, 6:1285-1292 (1991); Chang et al., Mol. Cell. Biol., 12:800-810, (1992)). This translocation results in a fusion between the retinoic acid receptor gene (RAR.alpha.) and a gene for a putative transcription factor, PML. The fusion product, PML-RAR.alpha., displays altered transactivating properties compared with wildtype RAR.alpha. gene product, which acts as a transcription enhancer in response to retinoic acid (RA) (Kakizuka et al., Cell, 66:663-674, (1991); de The et al., Cell, 66:675-684 (1991); Pandolfi et al., Oncogene, 6:1285-1292 (1991)). It has been shown that ATRA induces maturation of the leukemia cells both in vivo (Varrell et al., New England J. Med., 329:177-189, (1991)) and in cultured cells (Lanotte et al., Blood, 77:1080-1086, (1991)), explaining the clinical effect of retinoids. This retinoic acid (RA)-responsiveness is tightly linked to the presence of the PML-RAR.alpha. gene product (Lanotte et al., Blood, 77:1080-1086, (1991); Miller et al., Proc. Natl. Acad. Sci. USA, 89:2694-2698 (1992)). From these and other findings (Grignani et al., Cell, 74:423-431 (1993)), it is postulated that PML-RAR.alpha. functions as a dominant negative mutation, its product blocking myeloid differentiation. Evidence for the involvement of the PML-RAR.alpha. protein in the pathogenesis of APL is provided by its expression in U937 cells, which results in a block in differentiation, increased sensitivity to RA, and increased cell survival in the presence of limiting serum in the culture media (Grignani et al., Cell, 74:423-431 (1993)).
Virtually all the APL patients display immature promyelocytes with the previously mentioned t(15;17) translocation. The precise location of this translocation at the molecular level is important, because different sequences are generated at the fusion junctions. Studies of a series of APL patients have shown that there is a large degree of heterogeneity among the various PML-RAR.alpha. transcripts (Miller et al., Proc. Natl. Acad. Sci USA, 89:2694-2698 (1992); Pandolfi et al., EMBO J., 11:1397-1407 (1992)), There are three sources of variability: (1) alternative splicing on the PML side of the mRNA, (2) alternative polyadenylation sites on the PML-RAR.alpha. side (3' end of the transcript) and (3) variable fusion points. Studies of a large number of APL cases have shown that the breakpoint in chromosome 17 is always located inside intron 2 of the RAR.alpha. sequence (Miller et al., Proc. Natl. Acad. Sci USA, 89:2694-2698 (1992); Pandolfi et al., EMBO J., 11:1397-1407 (1992)). This results in the presence of the same RAR.alpha. sequence in all the variants of PML-RAR.alpha. transcripts. Breakpoints in chromosome 15, on the PML gene are instead clustered in three different regions, defined as bcrl, bcr2 and bcr3 (Pandolfi et al., EMBO J., 11:1397-1407 (1992)). The bcr1 region spans the whole length of intron 6 of the PML gene, and translocations involving this breakpoint result in the generation of a mature mRNA in which exon 6 of PML and exon 3 of RAR.alpha. are spliced together. The bcr2 region spans a region encompassing a small portion of intron 4, exon 5, intron 5 and exon 6 of PML. Translocations involving this breakpoint are essentially different from one another and many of them occur inside PML exons, causing a large variation in the fusion sequences and, occasionally, generating aberrant reading frames, which code for aberrant and truncated proteins. The bcr3 region is located in intron 3 of PML and invariably results in a mRNA in which exon 3 of PML and exon 3 of RAR.alpha. are spliced together. The sequence in the fusion junction is identical in all the bcr3 cases. Taken together, bcrl and bcr3-type junctions account for at least 80 percent of the tested APL cases (Pandolfi et al., EMBO J., 11:1397-1407 (1992)), with one study finding bcr1-type junctions at twice the rate of bcr3-type ones (Miller et al., Proc. Natl. Acad. Sci USA, 89:694-2698 (1992)).
Other Translocational Cancers
Many other cancers have been reported in the literature as arising due to, or associated with, chromosomal translocations. Examples include RBTN2 and t[11; 14] [p13; q11] in T cell acute leukemia and erythropoiesis, translin in lymphoid neoplasms, T[5;14][q34;q11] in acute lymphoblastic leukemia, T14;18 chromosomal translocations in follicular lymphoma, Non-Hodgkin's lymphomia, Hodgkin's disease; T18 translocations in human synovial sarcomas; Burkitt's lymphoma; t[11; 22] [q24; q12] translocation in Ewing sarcoma; t[3p; 6p] and t[12q; 17p] translocations in human small cell lung carcinomas; and t[15; 19] translocation in disseminated mediastinal carcinoma. In many of these cases, the transcription product of the fusion or the fusion itself represent targets for therapy, if a therapeutic agent could be designed which would selectively kill or inactivate those cells having the translocation.
It is therefore an object of the present invention to provide a therapeutic targeted for treatment of viral diseases and diseases involving abnormal transcription products, and method of use thereof.
It is another object of the present invention to provide modified external guide sequences for RNAse P with enhanced resistance to nuclease degradation.
It is another object of the present invention to provide methods of cleaving target RNA molecules mediated by modified external guide sequences for RNAse P.
It is a further object of the present invention to provide an external guide sequence for RNAse P specifically targeted against hepatitis, vectors encoding such external guide sequences, and methods of use thereof.